The long term objectives are: a) to understand the nature of the alterations which render enzyme molecules inactive in cells of senescent organisms; b) to study the causes of the age-associated accumulation of altered molecules; and c) to study the physiological consequences of reduced enzyme activitics and the accumulation of altered enzyme molecules on the adaptive capacity of cells to respond to environmental challenges. Specifically, the current aims are first to try to study the chemical nature of the alterations in the enzyme molecules. Secondly, to investigate the components of the intracellular protein degredation system which misfunction in old age and thus cause a slower disposal of aberrant protein molecules. Thirdly, to study the effect on erythrocyte function and longevity of the age-associated reduction in the specific activity of enzymes which scavenge reactive oxygen species. Inactive enzyme molecules of superoxide dismutase, aldolases A and B, and glyceraldhyde-3-dehydrogenase will be separated from active moleucles in tissue preparations of old animals. This separation will be carried out by a novel method using antibodies prepared against denatured enzyme molecules. The molecular weight, amino and carbosyl termini and proteolytic pattern after controlled digestion with Staphylococcus aureus protease will be determined. Amino acid analysis aimed at searching for altered cysteine, methionine, and histidine will be caried out. An analysis of denatured domains in the altered molecules will be carried out with monoclonal antibodies which will be developed against the denatured molecules or against selected peptides derived from their proteclysis. An in vitro system in cell homogenates will be developed which will yeild the same degradation intermediates as are obtained in vivo. Fractionation and suitable analysis of the homogenates will help in the identification of the proteases involved in intracellular protein degradation and which of them misfunction in aging cells. The life time of erythrocytes in the circulation will be related to their enzymatic capacity to withstand oxidative damage. Humoral vs intrinsic effects on their longevity will be tested by reciprocal transfer of erythrocytes between animals of various ages.